As the number of severe fungal infections continues to rise, the need for a broad spectrum antifungal agent becomes more urgent. The rise in fungal infections is primarily due to the increasing number of immuno-compromised patients as a result of medical advances (transplantation and chemotherapy) and as a result of the increasing population of AIDS patients.
Although fluconazole has been an effective drug against fungal pathogens for a number of years, resistance is increasing. Alternatives such as amphotericin B have serious drawbacks, including such side effects as nephrotoxicity and severe discomfort. Few new antifungals are on the horizon, and more knowledge about the pathogenicity of fungi as well as about their general biology is crucial if new drug targets are to be identified.
More than 80% of fungal infections in immuno-compromised patients are caused by Candida species. Cryptococcosis is the second most prevalent fungal infection in AIDS patients following candidiasis. Aspergillosis is responsible for at least 30% of the infections in cancer and organ transplant patients and has a high mortality rate.
In order to discover new drugs to combat fungal infections, compounds are often tested for their effects on particular, suitable polynucleotides and polynucleotide products. Suitable polynucleotides are generally those which are found to be essential to the viability of the pathogen. This determination of essentiality presents substantial obstacles to the identification of appropriate targets for drug screening. These obstacles are especially pronounced in diploid organisms, such as Candida albicans. 
A central technique used to investigate the role of a Candida albicans polynucleotide is to study the phenotype of a cell in which both copies of the polynucleotide have been deleted. Two popular polynucleotide deletion protocols have been reported. The first and most often used method, the ‘urablaster’ method, requires construction of a disruption cassette consisting of a selectable marker (URA and hisG flanking sequences) and sequences of the polynucleotide to be disrupted that are positioned at the 5′ and 3′ ends of the hisG-URA3-hisG cassette. A ura3− strain is then transformed and grown on minimal medium lacking uridine. ura3+ clones are isolated and transferred to a medium containing 5-fluoroortic acid, which selects for strains that have a ura3− genotype. These arise spontaneously as a subpopulation of the original transformed cells which will have undergone a recombinational event that retains one of the hisG sequences in the disrupted allele. The heterozygote ura3− strains can then be used in a second transformation event using the same cassette to disrupt the second allele resulting in a homozygous deletion strain.
A second type of popular method utilizes a PCR-based polynucleotide disruption strategy and multiple markers to construct homozygous mutants (Wilson et al J. Bacteriol. 181: 1868-74 (1999)). Although more rapid than the urablaster method, both methods have limitations. Since a homozygous deletion strain which lacks both essential polynucleotide copies would not be viable, such results are not an unequivocal explanation establishing the essential nature of the target polynucleotide because alternative explanations, including poor growth of a viable mutant strain, may be as likely a reason as essentiality for the negative results obtained.
Essential polynucleotides may also be identified using inducible promoter-regulated constructs to modify expression of the second polynucleotide copy, rather than completely inactivating it. With these methods, one polynucleotide copy is disrupted and the second copy is only expressed under certain conditions. The essentiality of the polynucleotide can be investigated since the fungal strain will only be viable under conditions in which the promoter is switched on (See, for example Nakayama et al. (Infection and Immunity 68: 6712-6719, (2000), and WO 01/60975).
One reportedly effective technique involves the use of the C. albicans MET3 promoter (Care et al., Molecular Microbiology 34 792-798 (1999)). The activity of the promoter is inhibited by methionine and/or cysteine and completely inactivated with both amino acids. Although the MET3 promoter is not the only regulated promoter to be characterized in C. albicans, one advantage of this promoter is that it is controlled by the addition of amino acids to the growth medium rather than a switch in carbon source. Switching carbon sources is likely to cause a bigger disturbance to cell physiology than adding amino acids (Care et at). However, the method developed for use of the MET3 promoter by Care et al. is cumbersome since it requires subcloning a portion of the polynucleotide under evaluation.
Additional methodologies enabling the identification of essential polynucleotides for drug screening which are both easy to use and give rapid results are needed, particularly for C. albicans. The C. albicans genome-sequencing project has recently begun and novel polynucleotides are being identified. There should be increasing demand to assess essential polynucleotide function and methodologies particularly amenable to high throughput screening will be useful.
Furthermore, although the identification of novel polynucleotides as essential in C. albicans is of value, the determination of essentiality for known polynucleotide sequences in C. albicans or other fungal genomes is also highly desirable since such polynucleotides will add to the set of drug targets. Furthermore, the use of C. albicans essential polynucleotides for drug screening for which orthologs in other pathogenic fungi are identified may result in the discovery of drugs effective in fighting infections from a variety of pathogens.